Measurement of infection efficiency of a major wheat pathogen using time-resolved imaging of disease progress

Infection efficiency is a key epidemiological parameter that determines the proportion of pathogen spores able to infect and cause lesions once they have landed on a susceptible plant tissue. In this study, an improved method to measure infection efficiency of Zymoseptoria tritici using a replicated greenhouse experiment is presented. Zymoseptoria tritici is a fungal pathogen that infects wheat leaves and causes septoria tritici blotch (STB), a major disease of wheat worldwide. A novel experimental setup was devised, where living wheat leaves were attached to metal plates, allowing for time-resolved imaging of disease progress in planta. Because lesions were continuously appearing, expanding and merging during the period of up to 3 weeks, daily measurements were necessary for accurate counting of lesions. Reference membranes were also used to characterize the density and spatial distribution of spores inoculated onto leaf surfaces. In this way, the relationship between the number of lesions and the number of viable spores deposited on the leaves was captured and an infection efficiency of about 4% was estimated from the slope of this relationship. This study provides a proof of principle for accurate and reliable measurement of infection efficiency of Z. tritici. The method opens opportunities for determining the genetic basis of the component of quantitative resistance that suppresses infection efficiency. This knowledge would improve breeding for quantitative resistance against STB, a control measure considered more durable than deployment of major resistance genes.     

猕猴桃软腐病主要病原菌的分离、鉴定及其生长特性研究

为探究猕猴桃果实采后软腐病害的主要病原菌及其生长特性,2017年9月于湖南凤凰县猕猴桃基地采集具有软腐病症状的100个果实病样,取患病猕猴桃病健交界处果肉,进行病原菌的分离纯化、菌株形态学观察、病原菌的分子生物学鉴定试验,同时分析不同培养基、碳源、氮源、温度和pH值对病原菌生长的影响。结果表明,菌种鉴定为葡萄座腔菌(Botryosphaeria dothidea)和间座壳菌(拟茎点霉菌有性态,Diaporthe phaseolorum)。两种菌在PSA、YPGA、PDA、SDA培养基的生长速度均较快,葡萄座腔菌和间座壳菌生长速率分别在16.1~18.6 mm·d^-1和13.9~16.5 mm·d^-1之间,且菌丝密度均较稠密。淀粉、蔗糖和麦芽糖作为碳源时,病原菌生长效果较好,葡萄座腔菌和间座壳菌生长速率分别在15.9~17.7 mm·d^-1和12.4~16.4 mm·d^-1之间。酵母粉、蛋白胨、甘氨酸作为氮源时,较适宜葡萄座腔菌和间座壳菌生长,两病原菌生长速率分别在11.0 mm·d^-1左右和9.5 mm·d^-1左右,但较未添加氮源培养基仅提高了7%左右。此外,葡萄座腔菌和间座壳菌的最适pH值为5~7,最适温度为25~30℃,菌种致死温度为85℃左右。本研究结果对猕猴桃软腐病病原菌的鉴定及对猕猴桃贮藏过程中软腐病害的绿色防控提供了参考。     

Formation of satellite uredinia as an important trait related to grapevine colonization by Phakopsora meliosmae-myrianthae

Phakopsora meliosmae-myrianthae, the causal agent of Asian grapevine leaf rust, significantly reduces the photosynthetic efficiency of grapevine leaves in green symptomless tissues surrounding lesions. This study took a close look at grapevine leaf colonization kinetics by P. meliosmae-myrianthae and compared it to P. pachyrhizi–soybean and Uromyces appendiculatus–bean colonization. It is already known from the literature that soybean rust, similar to grapevine rust, has a negative effect on leaf photosynthesis greater than would be expected based on visual lesions. However, in contrast to soybean and grapevine rusts, the effect of bean rust on leaf photosynthesis is proportional to the diseased leaf area. Colonization progress was monitored by fungal biomass assessed via histological staining and quantitative polymerase chain reaction (qPCR). Individual lesions of P. meliosmae-myrianthae on grapevine, P. pachyrhizi on soybean and U. appendiculatus on common bean leaves were evaluated every 3–4 days, and the number of uredinia was counted. Staining showed that mycelial colonization did not extend beyond the lesion border. The number of P. pachyrhizi and P. meliosmae-myrianthae uredinia within the lesions increased over time (on average 14-fold), whereas the number of U. appendiculatus uredinia remained the same. These findings were corroborated by qPCR, which revealed a greater increase in fungal biomass for Phakopsora spp. than for U. appendiculatus until 12 days post-inoculation. The high number of satellite uredinia within lesions might be directly related to the impact of this pathogen in photosynthetic efficiency on symptomless areas of diseased grapevine leaves. This study identified accelerated formation of satellite uredinia as an important feature of grapevine colonization by P. meliosmae-myrianthae.     

云南省珠芽魔芋(Amorphophallus bulbifer)软腐病病原菌鉴定

 魔芋 (Amorphophallus spp.) 是我国西南地区广泛种植的一种重要经济作物。2016~2018年在云南省发现一种由细菌侵染引起的珠芽魔芋新病害,症状表现为茎秆或叶片出现水渍状病斑,茎秆软化下垂,根部及球茎腐烂变褐,甚至整株倒伏。根据病害发病症状,并通过菌落形状、致病性测定、Biolog和16S rDNA测序分析,结果显示该病原菌在LB培养基上菌落呈近圆形,中央隆起,淡奶油色,质地均匀,表面光滑发亮;扫描和透射电子显微镜下观察显示,菌体形态呈短杆状,周生鞭毛,两端稍钝圆;基于16S rDNA扩增序列系统进化分析与已报道的Pectobacterium aroidearum strain CL1904-3 (MK875007) 相似性达99%以上,构建的系统进化树表明与P. aroidearum菌株亲缘关系最近,Biolog测定和16S rDNA序列分析均将该病原菌鉴定为软腐果胶杆菌 (P. aroidearum)。这是我国首次报道由P. aroidearum引起的珠芽魔芋软腐病害。研究结果进一步加深了人们对该病害的认识,为其防治提供了科学依据。     

Effects of deoxynivalenol exposure time and contamination levels on rainbow trout

The trend toward using plant‐based ingredients in aquafeeds is set to intensify; however, mycotoxin contamination might be a challenge. Two diets, with deoxynivalenol (DON) levels of 1,166 μg/kg (1.1 DON) and 2,745 μg/kg (2.7 DON), were prepared for short‐term DON exposure (50 days). A third diet with a low DON level of 367 μg/kg (0.3 DON) was prepared for long‐term DON exposure (168 days). Ingestion of DON by trout during both short‐term/high‐dosage exposure (50 days; 1,166 μg/kg and 2,700 μg/kg DON) and long‐term/low‐dosage exposure (168 days; 367 μg/kg DON) impacted growth performance and, to a lesser extent, liver enzyme parameters (2.7 DON). Histopathology showed mild to moderate changes in the liver but not in the other sampled tissues (intestine and kidney). Despite these effects, short‐term exposure of rainbow trout to high doses of DON did not result in increased susceptibility to Yersinia ruckeri. In both the short‐ and long‐term studies, the effects of DON showed a high interindividual variability. The present study confirms that subclinical levels of mycotoxins affect rainbow trout. The effects of such low mycotoxin levels could be masked by other production challenges while still negatively affecting productivity.     

广东省冬枣叶枯病病原菌的分离与鉴定

 2018年2月,广东省清远市冬枣种植区发生了一种叶枯病,发病率约为17%,严重影响植株生长。为明确引起广东省清远市冬枣叶枯病的病原,采用常规病组织分离法获得2株菌株,通过致病性、生理生化特征和分子生物学对2株菌株进行鉴定。致病性测定结果显示,该细菌可侵染引起冬枣叶片产生枯死症状,与田间病株的症状相同。生理生化测定结果显示,该病原菌为革兰氏阴性菌,在LB平板产生黄色菌落;除利用蜜二糖产酸结果不同,2株菌株与成团泛菌Pantoea agglomerans ATCC27155菌株的其他生理生化特征相同。系统进化分析结果表明,QYZ1和QYZ2 2株菌株的16S rDNA序列分别与P. agglomerans TH81 (CP031649)菌株、P. agglomerans C410P1 (CP016889)菌株、P. vagans FDAARGOS_160 (CP014129)菌株和P. vagans C9-1 (CP002206) 菌株的相似性在99%以上;多位点序列分析结果表明,QYZ1和QYZ2与P. agglomerans TH81菌株关系最近,聚在一个分支。生理生化特征及分子生物学鉴定结果表明,引起广东省冬枣叶枯病的病原为成团泛菌。本研究是成团泛菌引起广东省冬枣叶枯病在国内的首次报道。     

蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的分离与鉴定

为明确蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的种类,本研究对采集的炭疽病样本进行分离纯化培养,得到1株蜘蛛抱蛋炭疽病病原真菌YYL,采用针刺回接实验测定其致病性,并在形态学鉴定基础上,克隆其ITS、ACT、GAPDH和CAL片段进行多基因联合鉴定。结果表明,该病原菌具有较强致病性,其菌落、菌丝和分生孢子形态符合百合炭疽菌(Colletotrichum lilii)的特征,且与百合炭疽菌相似度最高,其ITS和ACT序列与百合炭疽菌完全一致。综合形态学特征和分子生物学鉴定结果,确定引起蜘蛛抱蛋炭疽病的病原菌为百合炭疽菌。     

Identifying the Pathogen of Cotton Leaf Spot Based on the Analysis of rDNA-ITS and Histone 3 Gene Sequence in Xinjiang

[Objective] The objective of this experiment is to identify pathogens causing cotton leaf spot in Xinjiang. [Method] Two hundred and six leaf samples displaying typical leaf spot symptoms were collected from 30 loci in 7 different regions of Xinjiang in 2016 and 2017. The fungal isolates were purified using conventional methods. Ten representative fungal strains were selected for further tests based on different sampling times, locations, and colony characteristics. The strains were identified based on their morphological characteristics and the sequence analysis of both rDNA internal transcribed spacer and histone 3 gene. [Result] In north of Xinjiang, Alternaria alternata and A. tenuissima were the dominant pathogens at the seedling stage (from May to June), whereas in August and September, A. tenuissima was the main pathogen and few were A. alternata and A. macrospora. In south of Xinjiang, A. tenuissima was the main pathogen at the seedling stage, whereas in August and September, A. tenuissima and A. macrospora dominated. Further, Gossypium hirsutum and G. barbadense was mainly infected with A. tenuissima and A. macrospora, respectively. [Conclusion] The main pathogens causing cotton leaf spot in Xinjiang are A. alternata, A. tenuissima and A. macrospora.     

Seed priming with Pseudomonas putida isolated from rhizosphere triggers innate resistance against Fusarium wilt in tomato through pathogenesis-related protein activation and phenylpropanoid pathway

Pathogenesis-related (PR) proteins are one of the major and preliminary proteins accumulated as a defense against biotic stress. This defense response can be induced by using beneficial rhizobacteria, which has been studied in various host-pathogen interactions. In the present study, eleven Pseudomonas isolates were assessed for their potential to ferment sorbitol, reduce nitrate, and produce mycolytic enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, phenazine antibiotics, and N-acyl homoserine lactones (AHLs). All isolates were tested against the host-specific pathogen Fusarium oxysporum MTCC1755 in tomato under greenhouse conditions, and shortlisted isolates were tested for their rhizosphere competence. In-vitro test results showed that the isolates were able to produce mycolytic enzymes, including protease, lipase, chitinase, cellulase, and amylase, and the antibiotic phenazine and were negative for pyoluteorin. All the isolates except two were positive for ACC deaminase production. Greenhouse results showed that the isolates M80, M96, and T109 significantly reduced symptoms of Fusarium wilt. Extended greenhouse tests under autoclaved and unautoclaved soil conditions showed that M80, M96, and T109 were excellent rhizosphere competitors and were identified as Pseudomonas putida. In brief, the defense-specific biochemical variations in the host could describe the improved defense against Fusarium wilt occurring in the primed plants. These three Pseudomonas strains could be used as potential biocontrol agents, along with their rhizosphere competence.     

刘家峡网箱鲟鱼鱼苗主要病原菌的分离鉴定

为查明夏季甘肃省永靖县刘家峡水库西伯利亚鲟鱼鱼苗爆发性疾病病原菌,以无菌方法从甘肃省永靖县刘家峡不同鲟鱼网箱养殖场共采集了30份濒死期及死亡鲟鱼的组织样品,采用常规细菌分离、分子生物学鉴定方法对病原菌进行了分离鉴定,并进行了人工感染实验和药敏实验。结果表明:从采集到的鲟鱼组织样品中分离到57株细菌,分别为铜绿假单胞菌、异常嗜糖气单胞菌、弗氏柠檬酸杆菌、河生莱略特氏菌、枯草芽胞杆菌、嗜水气单胞菌、鲁氏耶尔森菌、迟钝爱德华氏菌和海豚链球菌9种,除枯草芽胞杆菌及迟钝爱德华氏菌外,其余7种细菌对健康鲟鱼均具有不同程度的致病性;药敏实验分离到的7种病原菌均对诺氟沙星、氧氟沙星、环丙沙星、哌拉西林、庆大霉素、氯霉素等抗生素高度敏感,对青霉素、万古霉素、克林霉素、苯唑西林等不敏感,甚至完全耐药。该研究初步查明了甘肃省永靖县刘家峡西伯利亚鲟鱼鱼苗夏季爆发性疾病的病原菌种类,为该地区鲟鱼疾病的预防和合理用药提供了理论依据。